CD8 positive T-lymphocytes play an important role in host immunity to viruses. Anti-viral CD8+ T-cells recognize MHC class I molecules bound to peptides derived from a cytosolic pool of viral proteins. Concerning the delivery of antigenic peptides to MHC class I molecules, our studies have addressed the following specific questions. First, there are two sources of antigenic peptides, native proteins and everything else. We have termed all the non-native sources, DRiPs, or defective ribosomal products and rapidly degraded proteins. DRiPs consist of prematurely terminated polypeptides and misfolded polypeptides produced from translation products of bona fide mRNAs in the proper reading frame. We have been analyzing the contribution of DRiPs to antigen presentation in viral infections. Second this project will examine the nature of the substrate from which antigenic peptides are derived and their contribution to antigen presentation. Third, these studies will examine potential mechanisms of delivering antigen to the class I processing pathway. This year we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. We have used RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.